Phytoremediation of TCE at Two Superfund Sites

Phytoremediation of TCE at Two Superfund Sites

Bioactive Zone

GW FlowGW Flow

Assessing Methanotroph Activity in the Rhizosphere

Angela S. Lindner, Ph.D.Environmental Engineering Sciences, University of Florida

Email: [email protected]

Personnel Related to This Project

• Graduate Research Assistant: – Ms. Adriana Pacheco, EES, UF

• Collaborators:– Dr. Donald Rockwood, Forestry, UF– Dr. Jud Isebrands, Environmental

Forestry Consultants, New London, WI– Dr. Robin Brigmon, Savannah River

Laboratory, Aiken, SC

• UF SBRP Directors:– Dr. Margaret James – Dr. Steve Roberts

Web page: www.superfund.ufl.edu

Lindner Group, UF, 2004

Overview• Background

– Phytoremediation– Description of

Superfund sites in this study

• Broad goals and specific aims

• Initial results• Current focus


Phytoremediation of Contaminants

Focus of this studySource:


Two Superfund Sites

LaSalle,Illinois(LaSalleElectricUtilities)

Aiken,S.C.(SavannahRiverEcologyLabs, SRS)


Description of the Sites

LaSalle

• Former electric utility manufacturing capacitors and in operation between 1943-1982

• Tetrachloroethylene (PCE) and trichloroethylene (TCE) detected in groundwater

• Plant species used in PCE site:– PCE site: poplar (Populus spp.) in

native soil – TCE site: juvenile poplar (Populus

spp.), willow (Salix spp.) in PVC pipes

• TCE concentration range: 4-254 ppb

• PCE concentration range: 6-838 ppb

SRS

• Constructed in the 1950s to produce basic materials used in nuclear weapons production. A DOE site contracted to Westinghouse Savannah River Company LLC.

• Variety of contaminants, including tritium and plutonium. TCE plume in a seepage successional ecosystem.

• Plant species:– Loblolly pine (Pinus taeda) in

native soil (20 years old)

• TCE concentration range: 6-20 ppb– Dechlorination products also detected.


Broad Goals

• Assess the differences in methanotrophicpopulation diversity and activity potentials observed in rhizosphere samples from a variety of plant species in two U.S. regions

• Verify relationships between plant species and methanotrophic population diversity and activity by simulating bench-scale phytoremediation


Three Levels of Study in the SBRP Phytoremediation Project

Laboratory

Field

Pilot

Pure culture

Enrichments

Root

sam

ples

La S

alle,

IL

SRS, Aiken, SC

Specific Aims:

1. Microbial Community and Activity Analysis of Rhizosphere Samples

2. Laboratory Enrichments and Activity Measurements

3. Bench-scale Studies


Questions Addressed Here

• Specific Aim 1: Rhizosphere Samples – How do heterotrophic and methanotrophic counts and activity differ

• By plant type?• In roots and soil?• With nonvegetated controls?• With different moisture levels?

• Specific Aim 2: Enrichments and Pure Cultures– What effect do terpenes have on TCE biodegradation processes

involving methanotrophs? – What are the dominant methanotroph species?

7

2

34

56

CH3 10

1

CH38CH3 9

alpha-pinene


Sampling Protocol

Bottom layerAprox. 75 cm

Middle layer

Aprox. 45 cm

0 – 15 cm Top layer

15 – 30 cm

3 species: Loblolly pine (SRS)

Poplar (LaSalle) Willow (LaSalle)

2 seasons: Mid Summer Mid Winter

24 samples

(Treatment #1)

+ 24 samples

(Treatment #2) +

16 samples (Treatment #3)

4 composite soil depth samples x x =

Overall sample load: 64 samples


Sampling Schedule

LaSalle

2003 20052004

Jul Jul Feb/Mar

SRS

1 Year – Plant growth(time changes)

Season change

HIGH RootProduction

LOW RootProduction

Season change

LaSalle

2003 20052004

Jul Jul Feb/Mar

SRS


Season change

HIGH RootProduction

LOW RootProduction

Season change

2003 20052004

Jul Jul Feb/Mar

SRS


Season change

HIGH RootProduction

LOW RootProduction

Season change

Next sampling at both sites: February/March 2005

Benefits of Sampling Protocol:

Allows for determining effects of stage of plant growth, season, and plant

maintenance techniques on populations and activities

Comparison of both sites will provide insight into effects of climate, plant age,

site characteristics, and plant type.

July 2003 July 2004Lindner Group, UF, 2004

La Salle Site Sampling

TCE Site

PCE Site

N

3.82 ppb

Flowdirection

High[PCE] Low

[PCE]

High[TCE]

Low[TCE]

16.9 ppb

TCE

ND

PCE

2.6 ppb

ND

18.2 ppbND

254 ppb4.73 ppb

6.63 ppb 838 ppb

Monitoring WellsSept 2002

X

X

XGrass & Clovercover

X Control

X X

XX

X Sampled area

TCE Site

PCE Site

N

3.82 ppb

Flowdirection

High[PCE] Low

[PCE]

High[TCE]

Low[TCE]

16.9 ppb

TCE

ND

PCE

2.6 ppb

ND

18.2 ppbND

254 ppb4.73 ppb

6.63 ppb 838 ppb

Monitoring WellsSept 2002

X

X

XGrass & Clovercover

X Control

XX XX

XXXX

XX Sampled area

TCE Site

PCE Site


SRSSampling

Sampled area

Trichloroethylene plumes in C-Area

Twin Lakes Wetland

TCE concentrations: 6-20 ppb


Microbial Counts in 2003 La Salle Samples

Low [TCE] Willow S365

Soil depth (cm)

0-15

15-30

30-60

60-90

log

(cel

l cou

nt o

r MPN

) g-1

soi

l

0

1

2

3

4

5

6

7

8

9

10

CFU hts-soilMPN mts-soil

0-15

15-30

30-60

60-90 0-1

515

-3030

-6060

-90

High [TCE]Willow S365

Control

ND ND NDND

Control(grass cover)

0-15

15-30

30-60

60-90

0

1

2

3

4

5

6

7

8

9

10Low [PCE]

Soil depth (cm)

0-15

15-30

30-60

60-90lo

g (c

ell c

ount

or M

PN) g

-1 d

ry s

oil o

r fre

sh ro

ot

0

1

2

3

4

5

6

7

8

9

10

CFU hts-soil CFU hts-roots MPN mts-soilMPN mts-roots

High[PCE]

0-15

15-30

30-60

60-90

* *

TCE Site PCE Site


Microbial Counts in 2003 SRS Samples

T re e # 1

S o il d e p th (c m )

0-15

15-30

30-60

60-90

90-120

log

(cel

l cou

nt o

r MPN

) g-1

dry

soi

l

0

1

2

3

4

5

6

7

8

9

1 0

C F U h ts -s o ilM P N m ts -s o il

T re e # 2

0-15

15-30

30-60

60-90

90-120

T re e # 3

0-15

15-30

30-60

60-90

90-120

C o n tro l

0-15

15-30

30-60

60-90

90-120

0

1

2

3

4

5

6

7

8

9

1 0


Summary of Microbial Counts

Microbial populations counts Soil characteristics

Heterotrophs (CFU)

Methanotrophs (MPN) S

ite

Co

nta

min

an

t

Soil (g-1 dry soil)

Root (g-1 fresh root)

Soil (g-1 dry soil)

Root (g-1 fresh root)

Water content

(%) pH

[TCE] or

[PCE] (ppb)

TC

E

108 - 109 (108) 109 - 1010 105 - 106

(105 - 106) 104 - 107

2 - 20

(11 - 21) 7 - 8(7 - 8) 4 - 254

LaS

alle

PC

E

106 - 108 (105 - 106) 108 - 109 104 - 106

(102 - 106) 103 - 105 9 - 33 (9 - 17)

6 - 8(6 - 7) ND - 838

SR

S

TC

E

106 - 109 (106) NA 101 - 104

(103 - 105) NA 12 – 100 4 - 5

(5) 6 - 20 (12-17)


Specific Aim 2• Impacts of alpha-pinene on TCE oxidation by

pure methanotrophs:

– Type I: Methylobacter album BG8– Type II: Methylosinus trichosporium OB3b– Type X: Methylococcus capsulatus (Bath)

• Activity and initial phylogenetic characterization of methanotrophic enrichments from field samples

H3C CH39 8

alpha-pinene1

2

47

35

6

H3C10


Known Environmental Pathways of TCE: Aerobic Conditions

ClCl

HCl

TCEClCl

HCl

TCEClCl

HCl

TCEsMMO

tol-2-MO tol DO

O

O

Cl

Cl

Cl

HH

H

chloral hydrate

ClCl

Cl

O

H

TCE epoxide

O

O-

O

H O-

O

+

glyoxalate formate

formate dehydrogenase

CO2

O

O-

Cl

Cl

ClO

Cl

Cl

Cl HH

H

O

O-

Cl

ClH CO

O

O-

O

H+ + + O

-O

H

trichloroacetate trichloroethanol dichloroacetate

glyoxalate formate

sMMOdehydrogenase

tol-2-MO

•Most methanotrophscan express particulate methane monooxygenase(pMMO).

•Some methanotrophscan also express soluble methane monooxygenase(sMMO).

•pMMO and sMMO can oxidize TCE.


Pure Culture Studies:Oxygen Uptake Analysis to

Measure the Effect of α-Pinene on TCE Oxidation

Norm

alized O2 Uptake R

ate

0.0

0.1

0.2

0.3

0.4

0.5

0.6TCE TCE + (R)A-Pinene

(R)A-Pinene(R)A-Pinene + TCE

NA

Time (s)

0 200 400 600 800 1000 1200

% D

O

40

50

60

70

80

90

100

110

cells A-PineneTCE

Norm

alized O2 Uptake R

ate

0.0

0.1

0.2

0.3

0.4

0.5

0.6TCE TCE + (R)A-Pinene

(R)A-Pinene(R)A-Pinene + TCE

NA

Time (s)

0 200 400 600 800 1000 1200

% D

O

40

50

60

70

80

90

100

110

cells A-PineneTCE

Figure 1. Diagram of oxygen uptake reactorFigure 1. Diagram of oxygen uptake reactor

% DO


Methanotrophs +

1. TCE alone2. α-Pinene alone3. TCE + α-Pinene

Ra t e

s are normalizedto methane to produce a dimensionless measure of oxygen uptake.

Pure Culture Studies: Effect of alpha-Pinene

on TCE Oxidation

A

TCE and (R)-α-pinene concentrations (ppm)

0 20 40 60Nor

mal

ized

rate

of o

xyge

n up

take

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

OB3b sMMO TCE vs Col 2 OB3b sMMO TCE vs Col 5

B

0 20 40 60

C

0 20 40 60

D

0 20 40 60

E

0 20 40 60

A

α

0 20 40 60Cha

nge

in th

e no

rmal

ized

oxy

gen

upta

ke

rate

in th

e pr

esen

ce o

f 20

ppm

TC

E

-0.09

-0.06

-0.03

0.00

0.03

0.06

0.09 B

0 20 40 60

C

0 20 40 60

D

0 20 40 60

E

0 20 40 60

IncreaseD

ecrease

(R)- -pinene concentration (ppm)

Pure methanotrophs withalpha-pinene and TCE separately

Effect of alpha-pinene on oxygenuptake rates at 20 ppm TCE

KEY:A, B: M. trichosporium OB3b cultured without and with copper, respectively. C, D: M. capsulatus [Bath] cultured without and with copper, respectively. E: M. album BG8 cultured with copper.● TCE○ R-α-pinene

Pacheco and Lindner. 2004. BECT. In press.

Description of Enriched Mixed Cultures

• Stable mixed methanotrophic-heterotrophic cultures were isolated from the rhizosphere of poplar (LaSalle), willow (LaSalle), and loblolly pine (SRS) trees.

• All mixed cultures can use methane as the only carbon source and exhibit sMMO activity when cultured without copper.

• The LaSalle cultures were shown to be capable of oxidizing TCE and alpha-pinene.

• All three cultures exhibited methane oxidation activity…..


Activity of LaSalle Enrichments in the Presence of CH4

TCE Zone: Cultures isolated from rhizosphere soil samples of all tree types

• PCE Site

Plant type

Type II-OB3b Control/Grass Poplar

Oxy

gen

upta

ke ra

te (u

mol

/s)

0.00

0.01

0.02

0.03

0.04

0.05

0.06

Plant type

Type II-OB3b Poplar Willow Loblolly pine

Oxy

gen

upta

ke ra

te (u

mol

/s)

0.00

0.01

0.02

0.03

0.04

0.05

0.06NMS medium (sMMO)NMS w/Cu medium (pMMO)

NA

Root Soil

Oxy

gen

upta

ke ra

te (u

mol

/s)

0.000

0.005

0.010

0.015

0.020

0.025

0.030Low [TCE]High [TCE]

(Control)

A

B

Oxy

gen

upta

ke ra

te (u

mol

/s)

0.00

0.01

0.02

0.03

0.04

0.05

0.06

Plant type

Type II-OB3b Poplar Willow Loblolly pine

Oxy

gen

upta

ke ra

te (u

mol

/s)

0.00

0.01

0.02

0.03

0.04

0.05


NA

Root Soil

Oxy

gen

upta

ke ra

te (u

mol

/s)

0.000

0.005

0.010

0.015

0.020

0.025

0.030Low [TCE]High [TCE]

(Control)

A

B

Oxy

gen

upta

ke ra

te (u

mol

/s)

0.00

0.01

0.02

0.03

0.04

0.05


Root Soil

Oxy

gen

upta

ke ra

te (u

mol

/s)

0.000

0.005

0.010

0.015

0.020

0.025

0.030Low [PCE]High [PCE]

A

B

(Control)

PCE Zone:Culture isolated from rhizosphere soil samples of poplar trees

Plant type

Type II-OB3b Poplar

NMS medium (sMMO)NMS w/Cu medium (pMMO)

Root Soil

Oxy

gen

upta

ke ra

te (u

mol

/s)

0.000

0.005

0.010

0.015

0.020

0.025


NA

A

B

(Control)Plant type

Type II-OB3b Poplar

NMS medium (sMMO)NMS w/Cu medium (pMMO)

Root Soil

Oxy

gen

upta

ke ra

te (u

mol

/s)

0.000

0.005

0.010

0.015

0.020

0.025


NA

A

B

(Control)

Cultures isolated from poplar soil and root samples

Activity of SRS Enrichments in the Presence of CH4

Plant type

Type II-OB3b Control Pine 1 Pine 3

Oxy

gen

upta

ke ra

te (u

mol

/s)

0.00

0.01

0.02

0.03

0.04

0.05

0.06

NMS (sMMO)NMS w/Cu (pMMO)


Development of PhylogeneticAnalysis of Rhizosphere Enrichments

from LaSalle/TCE Site

• Based on the nucleotide sequence of the pmoA geneobtained from a rhizosphereroot sample

• Length of branches is proportional to % dissimilarity (0.1 base changes per nucleotide sequence position)

• Rhizosphere samples placed in a separated branch (high % dissimilarity)

• Closest branch to rhizospheresamples dominated by uncultured bacteria from peatsoils and soda lake sediments

0.1

Uncultured bacterium RB-16 (AY236517.1)

Uncultured bacterium SL4.121 (AY550719.1)

Uncultured bacterium SL.1.15 (AY550689.1)51


Uncultured bacterium MLM52 (AY572002.1)

Uncultured bacterium MLM46 (AY572001.1)73

29



100

Uncultured bacterium A55 (AY488061.1)

Rhizosphere sample #1



10049

63

Methanotrophic bacterium LK5 (AF533663.1)

Uncultured bacterium LOPB13.2 (AF358052.1)

Uncultured bacterium Maahas2 (AJ430797.1)

Methylobacter sp. BB5.1 (AF016982.1)

Uncultured bacterium 5hm22 (AY236085.1)

Uncultured bacterium 1HM25 (AY550699.1)




62

81

47

100

73

35

74

100

89


Uncultured bacterium SL1.119 (AY550708.1)42

42

0.1



Uncultured bacterium SL.1.15 (AY550689.1)51




29



100

Uncultured bacterium A55 (AY488061.1)




10049

63

Methanotrophic bacterium LK5 (AF533663.1)

Uncultured bacterium LOPB13.2 (AF358052.1)

Uncultured bacterium Maahas2 (AJ430797.1)

Methylobacter sp. BB5.1 (AF016982.1)

Uncultured bacterium 5hm22 (AY236085.1)

Uncultured bacterium 1HM25 (AY550699.1)




62

81

47

100

73

35

74

100

89


Uncultured bacterium SL1.119 (AY550708.1)42

42


Conclusions to Date• Specific Aim 1

– Results from the field have shown that willows, loblolly pine, and certain clones of poplars have been shown to uptake PCE and TCE at both sites.

– Heterotroph and methanotroph counts in the 2003 root and soil samples seem to be impacted most by soil moisture content, soil/root location, and possibly tree type.

• Effects of age and tree growth at both sites will be determined by comparing 2004 counts to the 2003 counts.

• Specific Aim 2– alpha-Pinene impacts TCE oxidation; however, with different effects

depending on the methanotroph type.– Mixed cultures isolated from the roots and soil of each tree type are capable

of oxidizing methane, TCE, and alpha-pinene. Differences in oxidation potential exist depending on rhizosphere origin (soil or root), tree type, and MMO expression.

• These initial results emphasize the importance of characterizing each phytoremediation site for type of methanotrophs present, bioavailablecopper concentrations, and contaminant oxidation potentials.


Current Focus– Specific Aim 1:

• Counts in one-year samples and next round of sampling• Soil testing for determining bioavailable nutrients• Protocol development

– phylogenetic analysis for methanotrophic diversity in soil and root samples

– stable-isotope probing (14CH4) of samples for methanotrophic activity

– Specific Aim 2:• Protocol development for analysis for products of alpha-pinene and

TCE oxidation by pure methanotrophs


Phytoremediation of TCE at Two Superfund Sites

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